During the last decades TB has evolved from a predominantly pulmonary infection into a multifaceted pathology with a growing rate of extrapulmonary cases. Until to date effective TB prevention programs are hampered by the absence of a rapid and field adapted screening assay. In high-income countries mycobacterial culture remains the diagnostic standard, but it is time-consuming and relatively expensive. Ideally, sputum microscopy based on three sputum smears can identify up to 67% of culture positive cases. HIV co-infection has been reported to impair the demonstration of Mycobacterium tuberculosis in sputa, although some investigators do not report any influence of the HIV serostatus on the AFB diagnosis. The higher percentage of extrapulmonary TB in HIV positive TB patients additionally increases the rate of AFB-negative TB cases. This renders tuberculosis an increasing diagnostic challenge and underlines an urgent need for improved laboratory tools for its diagnosis.
Current approaches for diagnosing TB are not satisfactory. The sputum test for pulmonary TB is not always effective, particularly if there are no detectable bacteria in the sputum, or no sputum sample can be obtained. In addition, this diagnostic test requires microscopy and/or culture of the bacteria to confirm the diagnosis, neither of which is especially suitable to diagnosis in the field. cerebrospinal fluid for diagnosis of TB-meningitis is also problematic, particularly in the field since, once again, microscopy and/or culture of the bacteria and/or an ELISA test is usually required to confirm the diagnosis.
Blood tests for TB are also known, but have a poor track record, being complex and unreliable. Urine tests are simpler and more reliable, but current methods require processing of the urine before performing the diagnostic test—such processing usually involving concentration of the urine.
Among the newly developed methods antibody tests against a number of mycobacterial antigens have been developed, but none of these tests has so far reached the needed specificity for routine diagnostic purpose. The drop of sensitivity in HIV positive cases is also a major constraint. A different approach is to measure immune responses to Mycobacterium tuberculosis specific antigens like ESAT-6, but so far the differentiation between latent TB infection and TB disease is not possible.
Tuberculosis is an extremely complex pathology existing in multiple forms, but always starting as an airborne infection. Pulmonary tuberculosis occurs immediately at the entry point of the microorganism and extrapulmonary tuberculosis is the result of further penetration into the body of the patient with the most widespread examples of tuberculous meningitis and bone tuberculosis. Complexity of the pathology determines multitude of various approaches tried during this century of modern medicine. Furthermore clinical and radiographic manifestations of HIV-related pulmonary tuberculosis are dramatically altered by immunodeficiency. These factors severely limit our capability of early symptomatic recognition of tuberculosis in HIV/TB patients and also increase the danger of TB transmission to relatives and caregivers of such patients.
Mycobacteria can potentially be recovered from a variety of clinical specimens, including upper respiratory collections (sputum, bronchial washes, bronchoalveolar lavage, bronchial biopsies and such); urine, feces, blood, cerebrospinal fluid (CSF), tissue biopsies, and deep needle aspirations of virtually any tissue or organ. Bacterial culture remains the gold standard in the diagnosis of tuberculosis, but it can take up to 6-8 weeks to make a conclusive diagnosis. There are three major technologies used for rapid (faster than bacterial culture) diagnosis of the mycobacterial infections:                Direct microscopy of sputum smears;        PCR-based assays;        Immunodiagnostic methods.        
Direct microscopy of sputum smears. More than a century ago, Robert Koch identified the etiologic agent of tuberculosis by staining it and culturing it from clinical specimens. Today, the diagnosis of tuberculosis is usually established using staining and culturing techniques that do not differ substantially from those that Koch used. Direct microscopy of sputum is the norm for the diagnosis of tuberculosis in developing countries today and it is the benchmark against which the efficiency of any new test must be assessed. It is applied to pulmonary tuberculosis, but is not very useful for children or for patients with initial stages of pulmonary tuberculosis.
PCR-based assays. A comparative study of the performance of PCR tests in seven laboratories has shown high levels of false-positive PCR-results, ranging from 3% to 20% (with an extreme of 77% in one laboratory). This relatively poor performance resulted from lack of monitoring of each step of the procedure and underscores the necessity for careful quality control during all stages of the assay.
Immunodiagnostic Methods.
The Tuberculosis Skin Test. This is the probably oldest immunological test for tuberculosis. A small amount of substance called PPD Tuberculin is placed just under the top layer of the skin on the forearm with a small needle. The test is read 48 to 72 hours after it has been given. Generally, a swelling of 10 mm. or more is considered positive. Many developing countries use BCG vaccination to protect against TB. After BCG vaccination, the PPD skin test usually becomes positive. Results of the skin test vary dependent on the quality of the PPD antigen, reactivity of the immune system and probably even race of the individual. This test also does not provide an unequivocal indication about the stage and location of the infection.
Serological tests for M. tuberculosis. This approach, based on the detection of antibody immune response to mycobacterial antigens is one of the most widely used in research and clinical environments. All serological tests have approximately the same sensitivity and specificity if they use purified antigens. The sensitivity of the best tests is in a range of 80% for smear-positive cases and 60-70% for smear negative cases. The reported specificity is generally high and is in a range of 95-100%.
Currently existing technologies are limited in their performance in several ways.
Most widely accepted rapid microscopic test requires several hours to complete, skillful technician and clinical laboratory environment. Test interpretation is far too difficult compared to current standards of rapid POC (point of care) testing in the infectious diseases area. Real cost of one analysis per one patient runs in the range of $100-150 for a US hospital. Clinical specificity of the test is very good, but any improvements in sensitivity will be more than welcome.
Skin test has sufficient sensitivity, but takes a long time and does not provide information about stage of pathological process and does not sufficiently differentiate infected and vaccinated individuals.
Serological tests usually do not have sufficient sensitivity. Test results vary with variations in the individual immune response to TB antigens. These tests practically do not work in HIV patients infected by M. tuberculosis. This factor severely limits their applicability in Africa and many Asian countries. In the US this group of patients constitutes the majority of TB infected patients as well.
PCR tests are widely used in developed countries, but are complex, expensive and are not sensitive enough to justify their use as a screening test in developing countries.
A preferred method for rapid diagnosis of infectious diseases is based on the detection of a bacterial antigen in the patient sample, that provides unequivocal proof of active infectious process caused by specific pathogen. The concept of using a direct antigen test for detection of mycobacterial infections was described in several publications.
For example, the development of one of the first direct antigen assay for M. tuberculosis was reported in 1982—a radioimmunoassay for the detection M. tuberculosis antigens in sputum of patients with active pulmonary tuberculosis, using a rabbit antibody specific to the whole cells of M. bovis (BCG vaccine). Autoclaved and sonicated sputum was used as a sample. The assay detected antigen in 38 of 39 sputum samples from patients with active tuberculosis pulmonary tuberculosis.
Later studies reported the development of the ELISA system for the detection of mycobacterial antigens in the cerebrospinal fluid of patients with tuberculous meningitis, also using antibodies specific to the whole cells of M. bovis. Both systems showed surprisingly high specificity. Despite the fact that LAM was the major antigen responsible for the detection, it was reported that M. kansasii showed 5% cross-reactivity, and M. intracellulare, M. avium, M. fortitum, and M. vaccae cross-reacted only at 2%. Others reported detection, by ELISA, of mycobacterial antigen in the CSF of nine of 12 patients with tuberculous meningitis, corresponding to the sensitivity of 81.25%. Specificity of the test was equal to 95%.
Practically all previous attempts to develop a test for diagnosis of tuberculosis have focused on the detection of the pulmonary form of the disease. Extrapulmonary forms, which are notoriously difficult to diagnose, attracted relatively little attention due to low prevalence rate compared to pulmonary forms. Until the 1950s and 1960s, extrapulmonary TB cases comprised only around 10% of all tuberculosis cases. The onset of the HIV/AIDS pandemic has changed the situation completely. These two diseases eventually merged into a new complex public health problem. Now fully 60% of untreated HIV patients develop active TB during their lifetime and up to 70% of TB patients are HIV infected in sub-Saharan Africa and Asia. Superimposition of HIV and TB changed not only the epidemiology of tuberculosis, but also the course of the disease itself. During the last decades TB has evolved from predominantly a pulmonary infection into a multifaceted pathology with an ever growing prevalence of extrapulmonary forms. It is estimated that extrapulmonary TB cases currently comprise up to 30% of all cases of tuberculosis; this number might even be an underestimation due to the lack of tools for rapid screening and diagnosis of extrapulmonary forms of tuberculosis. Moreover, even pulmonary tuberculosis in HIV patients frequently exhibits atypical symptoms. For example, such patients typically do not produce sputum. These factors severely limit our capability of early symptomatic recognition of tuberculosis in HIV/TB patients and also increase the danger of TB transmission to relatives and caregivers of such patients. An easy to use screening test, capable of detecting a broad spectrum of pathologies due to M. tuberculosis infection, is urgently needed, including a test for extrapulmonary forms of TB. Such a need has long been discussed with no progress towards realising goal. Today the need has became a public health care emergency.
In other cases of pulmonary bacterial infections, the current screening process of choice is based on the detection of polysaccharide antigens secreted in the patient's urine. Bacterial polysaccharides are composed of monosaccharides uncommon to humans and therefore resistant to cleavage by human enzymes. This enables their secretion in urine in immunochemically intact forms suitable for detection by a polysaccharide-specific immunoassay. Extremely low concentrations of bacterial polysaccharides secreted in urine require very high sensitivity of the immunoassay in order to use it as a screening procedure.
Collaborating research groups from Sweden and Norway attempted development of a LAM-specific ELISA system detecting LAM antigen in patient urine. The system used antigen capture for detecting tuberculosis from urine based on lipoarabinomannan, a polysaccharide present on the surface of Mycobacterium tuberculosis, the organism responsible for causing tuberculosis in humans, as disclosed in PCT application no. WO97/34149 to Svenson, hereby incorporated by reference herein. The disclosed diagnostic procedure detected the presence of LAM in patient urine in 81.3% of AFB-positive patients and 57.4% of AFB-negative patients and demonstrated utility of the detection of mycobacterial LAM antigen for diagnosis of mycobacterial infections. At the same time the system failed to demonstrate utility of the disclosed process for screening purposes. Despite use of the affinity purified rabbit polyclonal antibody specific to LAM antigen, the procedure lacked sufficient sensitivity to be used on non-processed un-concentrated urine samples. The diagnostic procedure required approximately 24-48 hrs of sophisticated manipulations in a biochemical lab focused on concentrating patient urine and preparing it for analysis by ELISA test. Overall, the sensitivity of the Svenson assay is not sufficient for practical use of the disclosed method. The complexity and length of the immunoassay also prevents its practical use as a screening test for detection of mycobacterial infections because it proved too cumbersome for use in a clinical setting, where speed, ease of use, and high sensitivity are all critically important for diagnostic tests used to detect disease conditions.